Vectors for use in filamentous fungi

ABSTRACT

Novel vectors are disclosed for use in filamentous fungi such as Aspergillus sp. in particular, whereby protein coding regions may be inserted therein to achieve expression or expression followed by secretion of the coded protein from the host. Signal peptide sequences and promoter sequences valuable for this purpose are disclosed as are expression vectors containing coding regions native or foreign to the fungal host. In accordance with the invention, a filamentous fungus such as Aspergillus may be provided with foreign or natural coding regions associated with foreign or natural promoter sequences and optionally signal peptide sequences which can be used to control the expression and/or secretion of the proteins encoded by these coding regions.

This is a Rule 60 continuation of application Ser. No. 08/237,405 filedMay 2, 1994 now U.S. Pat. No. 5,503,991, which is a continuation ofapplication Ser. No. 07/977,832, filed Nov. 17, 1992, now abandonedwhich is a continuation of application Ser. No. 06/811,404, filed Dec.20, 1985, now U.S. Pat. No. 5,198,345.

FIELD OF THE INVENTION

This invention relates to expression and expression followed bysecretion of proteins from filamentous fungi.

BACKGROUND OF THE INVENTION

One goal of recombinant DNA technology is the insertion of structuralgenes which encode commercially or scientifically valuable proteins intoa host cell which is readily and economically available. Genes selectedfor insertion are normally those which encode proteins produced in onlylimited amounts by their natural hosts or those which are indigenous tohosts too costly to maintain. Transfer of the genetic information in acontrolled manner to a host which is capable of producing the protein ineither greater yield or more economically in a similar yield provides amore desirable vehicle for protein production.

Genes encoding proteins contain promoter regions of DNA which areessentially attached to the 5' terminus of the protein coding region.The promoter regions contain the binding site for RNA polymerase II. RNApolymerase II effectively catalyses the assembly of the messenger RNAcomplementary to the appropriate DNA strand of the coding region. Inmost promoter regions, a nucleotide base sequence related to thesequence TATATA, known generally as a "TATA box" is present and isgenerally disposed some distance upstream from the start of the codingregion and is required for accurate initiation of transcription. Otherfeatures important or essential to the proper functioning and control ofthe coding region are also contained in the promoter region, upstream ofthe start of the coding region.

Filamentous fungi, particularly the filamentous ascomycetes such asAspergillus, e.g. Aspergillus niger, represent a class ofmicro-organisms suitable as recipients of foreign genes coding forvaluable proteins. Aspergillus niger and related species are currentlyused widely in the industrial production of enzymes e.g. for use in thefood industry. Their use is based on the secretory capacity of themicroorganism. Because they are well characterized and because of theirwide use and acceptance, there is both industrial and scientificincentive to provide genetically modified and enhanced cells of A. nigerand related species including A. nidulans, in order to obtain usefulproteins.

Expression and secretion of foreign proteins from filamentous fungi hasnot yet been achieved. It is by no means clear that the strategies whichnave been successful in yeast would be successful in filamentous fungisuch as Aspergillus. Evidence has shown that yeast is an unsuitablesystem for the expression of filamentous fungal genes (Pentilla et alMolec. Gen. Genet. (1984) 194:494-499) and that yeast genes do notexpress in filamentous fungi (F. Buxton personal communication). Geneticengineering techniques have only recently been developed for Aspergillusnidulans and Aspergillus niger. These techniques involve theincorporation of exogenously added genes into the Aspergillus genome ina form in which they are able to be expressed.

To date no foreign proteins have been expressed in and secreted fromfilamentous fungi using these techniques. This has been due to a lack ofsuitable expression vectors and their constituent components. Thesecomponents include Aspergillus promoter sequences described above, theregion encoding the desired product and the associated sequences whichmay be added to direct the desired product to the extracellular medium.

As noted, expression of the foreign gone by the host cell requires thepresence of a promoter region situated upstream of the region coding forthe protein. This promoter region is active in controlling transcriptionof the coding region with which it is associated, into messenger RNAwhich is ultimately translated into the desired protein product.Proteins so produced may be categorized into two classes on the basis oftheir destiny with respect to the host.

A first class of proteins is retained intracellularly. Extraction of thedesired protein, when intracellular, requires that the geneticallyengineered host be broken open or lysed in order to free the product foreventual purification. Intracellular production has several advantages.The protein product can be concentrated i.e, pelleted with the cellularmass, and if the product is labile under extracellular conditions orstructurally unable to be secreted, this is a desired method ofproduction and purification.

A second class of proteins are those which are secreted from the cell.In this case, purification is effected on the extracellular mediumrather than on the cell itself. The product can be extracted usingmethods such as affinity chromatography and continuous flow fermentationis possible. Also, certain products are more stable extracellularly andare benefited by extracellular purification. Experimental evidence hasshown that secretion of proteins in eukaryotes is almost always dictatedby a secretion signal peptide (hereafter called signal peptide) which isusually located at the amino terminus of the protein. Signal peptideshave characteristic distributions as described by G. Von Heijne in Eur.J. Biochem 17-21 (1983) and are recognizable by those skilled in theart. The signal peptide, when recognized by the cell, directs theprotein into the cell's secretory pathway. During secretion, the signalpeptide is cleaved off making the protein available for harvesting inits manure form from the extracellular medium.

Both classes of protein, intracellular and extracellular, are encoded bygenes which contain a promoter region coupled to a coding region. Genesencoding extracellularly directed proteins differ from those encodingintracellular proteins in that the portion of the coding region nearestto the promoter (which is the first part to be transcribed by RNApolymerase) encodes the signal peptide portion of the protein. Thenucleotide sequence encoding the signal peptide, hereafter denoted thesignal peptide coding region, is operationally part of the coding regionper se.

SUMMARY OF THE INVENTION

In the present invention, from one aspect, a promoter region associatedwith a coding region in filamentous fungus such as A. niger, A. nidulansor a related species is identified and isolated, appropriately joined ina functional relationship with a second different coding region, outsidethe cell, and then re-introduced into a host filamentous fungus using anappropriate vector. Then the host cells express the protein of thesecond coding region, under the control of the introduced promoterregion. The second coding region may be one which is foreign to the hostspecies, in which case the host will express and in some cases secrete aprotein not naturally expressed by the given host. Alternatively, thesecond coding region may be one which is natural to the host, in whichcase it is associated with a promoter region different from the promoterregion with which it naturally associates in the given host, to givemodified or enhanced protein expression and secretion.

In another aspect, where the second coding region encodes a proteinwhich is normally secreted then the second coding region itself willcontain a sequence of nucleotides at its 5' terminus i.e. a signalpeptide coding region, which will result, following transcription andtranslation, in the presence of a signal peptide at the amino terminusof the protein product. The signal peptide will be recognized by thefungal host and the protein product will then be directed into thesecretory pathway of the cell.

In another aspect, the present invention provides an appropriate DNAsequence coding for a signal peptide i.e. a signal peptide codingregion, which is recognized by filamentous fungi preferably of theascomycetes class so as to signal secretion of a protein encoded withinthe coding region. As indicated above, these signal peptide codingregions may be coupled to a coding region which encodes a proteinnaturally retained intracellularly, in order to elicit secretion of thatprotein. Normally secreted proteins are encoded by coding regions whichusually contain these signal peptide coding regions naturally so thatincorporation of a signal peptide coding region is not usuallynecessary. Nevertheless, the signal peptide coding regions of thepresent invention may be substituted for the naturally occurring suchsequence, if desired. Accordingly, where a signal peptide coding regionis inserted into a vector to obtain secretion, it will be foreign to thecoding region.

The present invention provides the ability to introduce foreign codingregions into filamentous fungi along with promoters to arrange for thehost fungi to express different proteins. It also provides the abilityto regulate transcription of the individual genes which occur naturallytherein or foreign genes introduced therein, via the promoter regionwhich has been introduced into the host along with the gene. Forexample, the promoter region naturally associated with the alcoholdehydrogenase I (alcA) gene and the aldehyde dehydrogenase (aldA) genesof A. nidulans are regulatable by means of ethanol, threonine, or otherinducing substances in the extracellular medium. This effect isdependent on the integrity of a gone known as alcR. When the alcA oraldA promoter region is associated with a different structural gene inAspergillus or the like, in accordance with the present invention,similar regulation of the expression of the different genes by ethanolor other inducers can be achieved.

As a further example, the promoter region naturally associated with theglucoamylase gone in Aspergillus niger and used in embodiments of thepresent invention is positively induced with starch and other sugars.

In another aspect, the present invention provides a DNA segment whichcontains a promoter region in operative association with a signalpeptide coding region and which permits introduction of a region codingfor a desired protein at a position 3' of and in reading frame with thesignal peptide coding region when the introduced coding region does notcontain a signal peptide coding region. The promoter/signal segment issuitably provided with a flanking restriction site to allow precisecoupling of the protein coding region to the signal peptide codingregion.

In another aspect, the present invention provides a genetic vectorcapable of introducing the segment carrying the promoter and signalpeptide coding region into the genome of a filamentous fungus host. Thevector may also include a protein coding region either native to orforeign to the host filamentous fungus.

Thus the present invention, provides DNA sequences active as promoterregions in association with coding regions in cells of filamentous fungisuch as Aspergillus niger, Aspergillus nidulans and the like.

The present invention thus also provides a novel composition of mattercomprising a DNA sequence active as a promoter region in cells offilamentous fungi, and a coding region chemically bound to said DNAsequence in operative association therewith, said coding region beingcapable of expression in a filamentous fungus host under influence ofsaid DNA sequence.

The present invention further provides a process of geneticallymodifying a filamentous fungus host cell which comprises introducinginto the host cell, by means of a suitable plasmid vector, a codingregion capable of expression in the Aspergillus host cell and a promoterregion active in the Aspergillus host cell, the coding region and thepromoter being chemically bound together and in operative associationwith one another.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preferred hosts according to the invention are the filamentous fungi ofthe ascomycete class, most preferably Aspergillus sp. including A.niger, A. nidulans and the like.

In the preferred form of the invention the promoter region associatedwith either the Aspergillus niger glucoamylase gene or the promoterregion associated with the alcohol dehydrogenase I gene or aldehydedehydrogenase gene associated with Aspergillus nidulans is obtained andused in preparing an appropriate vector plasmid.

Either or all of these promoter regions is regulatable in the host cellby the addition of the appropriate inducer substance. In alcA and aldA,this induction is mediated by a third gene, alcR which is controlled viathe promoter. Multiple copies of alcR may be used to increase theexpression of alcA, aldA and related sequences. In some instances thegene can be repressed, for example by utilizing high bevels of glucose,(and some other carbon sources) in the medium to be used for growth ofthe host. The expression of the product encoded by the coding region isthen delayed until after the end of the cell growth phase, when all ofthe glucose has been consumed and the gene is derepressed. The inducermay be added at this point to enhance the activity of the gene.

The destination of the protein product of the coding region which hasbeen selected to be expressed under the control of the promoterdescribed above is determined by the nucleotide sequence of that codingregion. As mentioned, if the protein product is naturally directed tothe extracellular environment, it will inherently contain a secretionsignal coding region. Protein products which are normallyintracellularly located lack this signal peptide.

Thus, for the purposes of the present disclosure it is to be understoodthat a "coding region" encodes a protein which is either retainedintracellularly or is secreted. (This "coding region" is sometimesreferred to in the art as a structural gene i.e. that portion of a genewhich encodes a protein.) Where the protein is retained within the cellthat produces it, the coding region will usually lack a signal peptidecoding region. Secretion of the protein encoded within the coding regioncan be a natural consequence of cell metabolism in which case the codingregion inherently contains a signal peptide coding region linkednaturally in translation reading frame with that segment of the codingregion which encodes the secreted protein. In the alternative, thecoding region may contain a signal peptide coding region which isforeign to that portion of the coding region which encodes the secretedprotein. This foreign signal peptide coding region may be required wherethe coding region does not naturally contain a signal peptide codingregion or it may simply replace the natural signal peptide coding regionin order to obtain enhanced secretion of the desired protein with whichthe natural signal peptide is normally associated.

In accordance with another preferred aspect of the invention, therefore,a signal peptide coding region is provided, if required i.e. when thecoding region which has been selected to be expressed under the controlof the promoter described above does not itself contain a signal peptidecoding region. The signal peptide coding region used is preferablyeither one which is associated with the Aspergillus niger glucoamylasegene or a synthetic signal peptide coding region which is made in vitroand used in the preparation of an appropriate vector plasmid. Mostpreferably, these signal peptide coding regions are modified at one orboth termini to permit ligation thereof with other components of avector. This ligation is effected in such a way that the signal peptidecoding region is interposed between the promoter region and the proteinencoding segment of the coding region such that the signal peptidecoding region is in frame with that segment of the coding region whichencodes the mature, functional protein.

BRIEF REFERENCE TO THE DRAWINGS

FIG. 1 is an illustration of the base sequence of the DNA constitutingthe structural genes and promoter regions of the alcohol dehydrogenase(alcA) gene (upper lines) and aldehyde dehydrogenase (ald A) gene (lowerlines) of Aspergillus nidulans.

FIG. 2 is a diagrammatic illustration of a process of constructing avector and of transforming a filamentous fungal cell therewith,according to the present invention;

FIG. 3 is a linear representation of a portion of the plasmid pDG6 ofFIG. 2;

FIG. 4 is a diagrammatic illustration of the plasmid maps of pGL1 andpGL2;

FIG. 5 (parts A-C) is an illustration of a selection of synthetic linkersequences for insertion into plasmid pGL2;

FIG. 6 is an illustration of the nucleotide sequence of a fragment ofpGL2;

FIG. 7 is an illustration of plasmid map pGL2B and pGL2BIFN;

FIG. 8 is an illustration of the nucleotide sequence of a fragment ofpGL2BIFN;

FIG. 9 illustrates plasmid pALCAlS and a method for its preparation;

FIG. 10 illustrates the plasmid map of pALCAlSIFN and a method for itspreparation;

FIG. 11 represents the nucleotide sequence of a fragment of pALCAlSIFN;

FIG. 12 illustrates the plasmid map of pGL2CENDO;

FIG. 13 represents the nucleotide sequence of a fragment of pGL2CENDO;

FIG. 14 represents a plasmid map of pALCAlSENDO;

FIG. 15 represents the nucleotide sequence of a fragment of pALCAlSENDO;

FIG. 16 illustrates plasmid pALCAlAMY and a method for its preparation;and

FIG. 17 represents the nucleotide sequence of a segment of pALCAlAMYshown in FIG. 16.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the present invention, an appropriate promoter region of afunctioning structural gene in A. niger or A. nidulans or the like isidentified. For this purpose, cells of the chosen species are induced toexpress the selected protein e.g. alcA and from these cells is isolatedthe messenger RNA. One portion thereof, as yet unidentified codes foralcA. Complementary DNA for the fragments is prepared from the mRNAfragments and cloned into a vector. Messenger RNA isolated from inducedA. nidulans was size fractionated to enrich for alcA sequences, endlabelled and hybridized to the cDNA clones made from the alcA⁺ strain.That clone containing the cDNA which hybridized to alcA⁺ mRNA Containsthe DNA copy of the alcA mRNA. This piece is hybridized to a total DNAgene bank from the chosen Aspergillus species, to isolate the selectedstructural gene e.g. alcA and its flanking regions. The aldA structuralgene was isolated using analogous procedures.

The coding region starts at its 5' end, with a codon ATG coding formethionine, in common with other structural genes and proteins. Wherethe amino acid sequence of the expressed protein is known, the DNAsequence of the coding region is readily recognizable. Immediately"upstream" of the ATG codon is the leader portion of the messenger RNApreceded by the promoter region.

With reference to FIG. 1, this shows portions of the total DNA sequencefrom A. nidulans, with conventional base notations. The portion shown inthe upper lines contains the promoter region and a part of the codingregion alcA encoding alcohol dehydrogenase I, and the portion shown inthe lower lines contains the promoter region and a part of thestructural gene encoding aldehyde dehydrogenase i.e. aldA. The aminoacid sequences of these two enzymes is known in other species. Fromthese, the region 10 is recognisable as the coding regions. Each startsat its 5' ("upstream") end with methionine codon ATG at 12, 12'. Theappropriate amino acid sequences encoded by the structural genes areentered below the respective rows on FIG. 1, in conventionalabbreviations. Immediately upstream of codon 12 is the messenger RNAleader and the promoter region associated with the coding region, thelength of which, in order to contain all the essential structuralfeatures enabling it to function as a promoter, now needs to bedetermined or at least estimated. FIG. 1 shows a sequence of 800 basesin each case, upstream from the ATG codon 12.

It is predictable from analogy with other known promoters that all thefunctional essentials are likely to be contained within a sequence ofabout 1000 bases in length, probably within the 800 base sequenceillustrated, and most likely within the first 200-300 base sequence,i.e. back to position 14 on FIG. 1. An essential function of a promoterregion is to provide a site for accurate initiation of transcription,which is known to be a TATA box sequence such as ⁵ ' TATATA³ '. Such asequence is found at 16 on the alcA promoter sequence of FIG. 1, and at16' on the aldA promoter sequence of FIG. 1. Another function of apromoter region is to provide, along with the coding region, anappropriate DNA sequence adjacent to the coding region sequence forregulation of the gene transcription, e.g. a binding site for aregulatory molecule which enhances gene transcription, or for renderingthe gene active or inactive. Such regulator regions are within thepromoter region illustrated in FIG. 1.

The precise upstream 5' terminus of the DNA sequence used herein as apromoter region is not critical, provided that it includes the essentialfunctional sequences as described herein. Excess DNA sequences beyondthe 5' terminus are unnecessary, but unlikely to be harmful in thepresent invention.

Having determined the extent of sequence containing all the essentialfunctional features to constitute a promoter region from the given gene,by techniques described herein, the next step is to cut the DNA chain ata convenient location downstream of the promoter region terminus and toremove the structural gene region, to leave basically a sequencecomprising the promoter region and sometimes part of the messenger RNAleader. For this purpose, appropriately positioned restriction sites areto be located, and then the DNA treated with the appropriate restrictionenzymes to effect scission. Restriction sites are recognizable from thesequences illustrated in FIG. 1. For the upstream cutting, a site ischosen sufficiently far upstream to include in the retained portion allof the essential functional sites for the promoter region. As regardsthe downstream scission, no restriction site presents itself exactly atthe ATG codon 12. The closest downstream restriction site thereto is thesequence GGGCCC at 22, at which the chain can be cut with restrictionenzyme Apa I. If desired, after such scission, the remaining nucleotidesfrom location 22 to location 12 can be removed, in stepwise fashion,using an exonuclease. With knowledge of the number of such nucleotidesto be removed, the exonuclease action can be appropriately stopped whenthe location 12 is passed. In many cases, however, a residue of aportion of the structural gene on the 5' terminus of the promoter regionis not harmful to and does not significantly interfere with thefunctioning of the promoter region, so long as the reading frame of thebase triplets is maintained.

FIG. 2 of the accompanying drawings illustrates diagrammatically thesteps in a process of preparing Aspergillus transformants according tothe present invention. On FIG. 2, 18 is a recombinant plasmid containingthe endogluconase (cellulase) coding region 30 from Cellulomonas fimi,namely a BamHI endoglucanase fragment from C. fimi in known vectorM13MPa. It contains relevant restriction sites for EcoRI Hind III andBamHI as shown as well as others not shown and not of consequence in thepresent process. Item 20 is a recombinant plasmid designated p5,constructed from known E. coli plasmid pBR322 and containing an EcORIfragment of A. nidulans containing the alcA promoter region prepared asdescribed above, along with a small portion of the alcA coding region,including the start codon ATG. It has restriction sites as illustrated,as well as other restriction sites not used in the present process andso not illustrated. Plasmid p5 contains a DNA sequence 22, from siteEcoRI (3') to site Hind III (5'), which is in fact a part of thesequence illustrated on FIG. 1, upper row, from base 240 (the sequenceGAATTC thereat constituting an ECoRI restriction site) to base -580thereon. Sequence 22 in plasmid 20 is approximately 2 kb in length.

The plasmids 18 and 20 are next cut with restriction enzymes EcoRI andHind III, and ligated to plasmid pUC12, so as to excise the alc Apromoter region and the endoglucanase (cellulase) gene and prepare anovel construct pDG5A containing these sequences on pUC12, as shown inFIG. 2. Plasmid pUC12 is a known, commercially available E coli plasmid,which replicates efficiently in E. coli, so that abundant copies ofpDG5A can be made if desired. Novel construct pDG5A is isolated from theother products of the construct preparation. Next, construct pDG5A isprovided with a selectable marker so that subsequently obtainedtransformants of Aspergillus into which the construct has successfullyentered can be selected and isolated. In the case of Arg B- Aspergillushosts, one can suitably use an Arg B gene from A. nidulans for thispurpose. The Arg B gene codes for the enzyme ornithine transcarbamylase,and strains containing this gene are readily selectable and isolatablefrom Arg B- strains by standard plating cut and cultivation techniques.Arg B- strains will not grow on a medium not containing arginine.

To incorporate a selectable marker, in this embodiment of the inventionas illustrated in FIG. 2, construct pDG5A may be ligated with the Xba Ifragment 32 of plasmid pDG3 (see U.S. patent application Ser. No.06/678,578 Buxton et al, filed Dec. 5, 1984) which contains the Arg B+gene from A. nidulans using Xba I, to form novel construct pDG6, whichcontains the endoglucanase coding region, the alCA promoter sequence andthe Arg B gene. Plasmid pDG6 can now be used in transformation, toprepare novel Aspergillus mutant strains containing an endoglucanasecoding region under the control of alcA promoter.

FIG. 3 shows in linear form the diagrammatic sequence of the functionalportion of construct pDG6, from the Hind III Site 24 to the Hind IIIsite 26. It contains the alcA promoter region, the ATG codon 12 and asmall residual portion 28 of the alcA structural gene 10 as shown inFIG. 1, followed by cellulase gene 30 derived from plasmid 18.

Plasmids pGL2 and pALCAlS as depicted in FIGS. 4 and 9 representparticularly preferred cloning vectors for introduction of proteincoding regions, promoter sequences and signal peptide coding regionsinto either A. niger or A. nidulans. These vectors provide forintroduction of entire coding regions which are foreign to these fungiand for introduction of protein encoding segments which are foreign toeither or both of the promoter region and signal peptide coding regionintegrated in these vectors.

Considering firstly the vector plasmid pGL2, reference is made to FIG. 4showing a map of the plasmid with relevant restriction sites indicatedthroughout the diagrams, the restriction sites illustrated comprisingthose sites which bear on the manner by which the plasmid or vector isproduced or may be manipulated according to the invention. Further sitesmay be present but are not considered relevant to the preferredembodiments shown and described herein.

pGL2 comprises the promoter region 20 of the glucoamylase gene native toA. niger the promoter region being situated between restriction sitesHind III and BssH II. Also comprised within region 20 is the geneticsequence coding for the signal sequence 22 of the glucoamylasestructural gene, the signal sequence 22 being located downstream of thepromoter 20. Cloned within the unique BssH II and Sst I sites i.e.downstream of the signal sequence 22 is a synthetic linker sequence 24designed so as to permit insertion of a desired structural gene withoutaltering the reading frame of the gene so inserted.

The synthetic linker component of pGL2 is preferably of one of thefollowing three nucleotide constructions denoted A, B or C as defined inFIGS. 5A, 5B and 5C, respectively.

Each of the synthetic linker sequences shown in FIGS. 5A, 5B and 5Cmakes available an Eco RV restriction site and a Bgl II/Xho IIrestriction site either of which may be used for insertion of thestructural gene. Moreover, it will be noted that all of the sequencesprovides for three reading frames as evidenced by the amino acidsequences appearing thereunder. The arg-ala amino acid sequence at thestart is constant in each oligonucleotide and is identical to the aminoacids flanking the signal peptide sequence cleavage Site in theglucoamylase gene. The addition of one, two or three "A" residues in thenucleotide sequence following arg-ala provides the three reading framesnecessary as shown by the variable amino acid sequence followingarg-ala. One advantage of such sequences resides in their capacity toaccept the structural gene as a component of the vector plasmid withoutaltering the reading frame of that gene thereby retaining, in theexpressed protein, the proper amino acid sequence.

Further, each synthetic linker sequence A, B and C is flanked by "stickyends" which are complementary to those ends provided by cuts of theplasmid pGL2 shown in FIG. 4.

The signal sequence 22 located upstream of the linker sequence 24 i.e.upstream of the BssH II site, has the nucleotide sequence as shownbelow:

    ______________________________________                                        Chart 1                                                                       ______________________________________                                        ATG TCG TTC CGA TCT CTA CTC GCC CTG AGC GGC CTC GTC                           TGC                                                                           met ser phe arg ser leu leu ala leu ser gly leu val cys                       ACA GGG TTG GCA AAT GTG ATT TCC AAG CGC                                       thr gly leu ala asn val ile ser lys arg                                       ______________________________________                                    

This signal sequence was excised from the glucoamylase gene indigenousto A. niger according to methods described hereinafter. It will be notedthat the restriction site for BssH II is located near the 3' terminus ofthe signal peptide sequence. It will be further noted that the 5'terminus of each of the synthetic linker sequences illustrated in FIGS.5A-5C provides a nucleotide sequence which provides a "sticky end" whichwhen ligated to the 3' end of the signal peptide coding region cut withthe same enzyme, results in a fragment in which the amino acid sequenceencoded by the signal peptide sequence is in frame with the codingregion chosen to be expressed and may function in its intended manneri.e. to direct secretion of the expressed protein encoding segment ofthe coding region which may be inserted at an appropriate position e.g.the Eco RV restriction site, within the synthetic linker 24. Thisdepends on the choice of the correct vector (pGLA,B or C).

The promoter region 20 of pGL2 is also derived from the glucoamylasegene indigenous to A. niger. In particular, a genetic fragmentcontaining both the promoter region and the signal peptide sequence forthe A. niger glucoamylase gene was excised from an appropriate genebank, as described hereinafter.

The invention is further described and illustrated by the followingspecific, non-limiting examples.

EXAMPLE 1

The vector construct pDG6 shown in FIG. 2 was prepared following theprocess scheme illustrated in FIG. 2, using standard routine ligationand restriction techniques. An EcoRI-Hind III fragment containing thepromoter region of the alcA gene, about 2,000 bases long was used. Withreference to the sequence on FIG. 1 upper row, it extends from base 240to 2,000 bases upstream, approximately. Then the construct pDG6 wasintroduced into Arg B- mutant cells of Aspergillus nidulans as follows:

500 mls of complete media (Cove 1966) +0.02% arginine +10⁻⁵ % biotin ina 2 l conical flask was innoculated with 10⁵ conidia/ml of an A.nidulans Arg B- strain and incubated at 30° C., shaking at 250 rpm for20 hours. The mycelia were harvested through Whatman No. 54 filterpaper, washed with sterile deionized water and sucked dry. The myceliawere added to 50 ml of filter sterile 1.2M MgSO₄ 10 mM potassiumphosphate pH 5.8 in a 250 ml flask to which was added 20 mg of Novozym234 (Novo Enzyme Industries), 0.1 ml (=15000 units) of β-glucuronidase(Sigma) and 3 mg of Bovine serum albumin for each gram of mycelia.Digestion was allowed to proceed at 37° C. with gentle shaking for 50-70minutes checking periodically for spheroplast production bylight-microscope. 50 mls of sterile deionised water was added and thespheroplasts were separated from undigested fragments by filteringthrough 30 um nylon mesh and harvested by centrifuging at 2500 g for 5minutes in a swing out rotor in 50 ml conical bottom tubes, at roomtemperature. The spheroplasts were washed, by resuspending andcentrifuging, twice in 10 mls of 0.6M KCl. The number of spheroplastswas determined using a hemocytometer and they were resuspended at afinal concentration of 10⁸ /ml in 1.2M Sorbitol, 10 mM Tris/HCl, 10 mMCaCl₂ pH 7.5. Aliguots of 0.4 ml were placed in plastic tubes to whichDNA pDG6 (total vol. 40 μl in 10 mM Tris/HCl l mM EDTA pH 8) was addedand incubated at room temperature for 25 minutes. 0.4 ml, 0.4 ml then1.6 ml aliguots of 60% PEG4000, 10 mM Tris/HCl, 10 mM CaCl₂ pH 7.5 wereadded to each tube sequentially with gentle, but thorough mixing betweeneach addition, followed by a further incubation at room temperature for20 minutes. The transformed spheroplasts were then added toappropriately supplemented minimal media 1% agar overlays, plus or minus0.6M KCl at 45° C. and poured immediately onto the identical (but cold)media in plates. After 3-5 days at 37° C. the number of colonies growingwas counted (F. Buxton et al), Gene. 37, 207-214 (1985)). The method ofYelton et al Proc. Nat'l Acad. Sci. U.S.A. 81; 1370-1374 (1980)! wasalso used.

The colonies were divided into two groups. Threonine (11.9 g/Liter) andfructose (1 g/Liter) were added to the incubation medium for one groupto induce the cellulase gene incorporated therein. No inducer was addedto the other group, which were repressed by growth on minimal media withglucose as sole carbon source. Both groups were assayed for generalprotein production by BioRad Assay, following cultivation, filtering toseparate the mycelia, freeze drying, grinding and protein extractionwith 20 mM Tris/HCl at pH 7.

To test for production of cellulase, plates of Agar medium containingcellulase (9 g/Lt, carboxymethylcellulose) were prepared, and smallpieces of glass fibre filter material, isolated from one another, and 75μg of total protein from one of the transconjugants was added to each ofthe filters. The plates were incubated overnight at 37° C. The filterswere then removed, and the plates stained with congo red to determinethe locations where cellulase had been present in the total protein onthe filters, as evidenced by the breakdown of cellulase in the agarmedium below. The plates were de-stained, by washing with 5M NaCl inwater, to detect the differences visibly.

Of four transformants induced with threonine and fructose, three clearlyshowed the presence of cellulase in the total protein product. Thenon-induced, glucose repressed transformants did not show evidence ofcellulase production.

Three control transformants were also prepared from the same vectorsystem and strains, but omitting the promoter sequence. None of themproduced cellulase, with or without inducers. The presence of C. fimiendoglucanase coding region was verified by the fact that medium fromthreonine-induced transformed strains showed reactivity with amonoclonal antibody raised against C. fimi endoglucanase. Thismonoclonal antibody showed no cross-reactivity with endogenous A.nidulans proteins in control strains.

EXAMPLE 2

The vector construct pALCAlAMY was prepared as indicated in FIG. 16,using standard routine ligation and restriction techniques. Inparticular, vector pALCAl 60 containing a Hind III-EcoRI segment inwhich the A. nidulans alcohol dehydrogenase 1 promoter 50 is located (asdescribed previously), was cut at its EcoRI site in order to insert thecoding region of the wheat α-amylase gene 80 contained within anEcoRI-ECoRI fragment defined by plasmid p501 (see S. J. Rothstein et al,Nature, 308, 662-665 (1984)). As wheat α-amylass is a naturally secretedprotein, its coding region 80 contains a signal peptide coding region 82and a segment 84 which encodes mature, secreted α-amylase. Ligation ofcoding region 80 within the EcoRI-cut site of pALCAl provides plasmidpALCA1AMY in which the AlcA promoter is operatively associated with theα-amylase coding region. The correct orientation of the p501-derivedα-amylase coding region within pALCA1AMY is confirmed by sequencingacross the ligation site according to standard procedures. Thenucleotide sequence of the promoter/coding region junction are shown inFIG. 17.

After transforming A. nidulans by the procedure described in example 1,samples of extracellular medium were taken from and applied to glassfibre filter papers placed on 1% soluble starch agar. The filters wereremoved after 8 hours at 37° C. and inverted onto beakers containingsolid iodine (in a 50° C. water bath). Clear patches indicated starchdegradation while the remaining starch turned a deep purple, thusconfirming the presence of secreted α-amylase.

EXAMPLE 3 Production of Plasmid pGL2

A) Source of promoter and signal peptide sequence

The glucoamylase gene of A. niger was isolated by probing a gene bankderived from DNA available in a strain of this microorganism on depositwith ATCC under catalogue number 22343. The probing was conducted usingoligonucleotide probes prepared with Biosearch oligonucleotide synthesisequipment and with knowledge of the published amino acid sequence of theglucoamylase protein. The amino acid sequence data was "reversetranslated" to nucleotide sequence data and the probes synthesized. Theparticular gene bank probed was a Sau 3A partial digest of the A. nigerDNA described above cloned into the Barn HI site of the commerciallyavailable plasmid pUC12 which is both viable in and replicable in E.coli.

A Hind III-Bgl II piece of DNA containing the glucoamylase gene wassubcloned into pUC12. Subsequently, the location of the desired promoterregion, signal peptide sequence and structural gene of glucoamylase wasidentified within pUC12 containing the sub-cloned fragment. The Eco RIfragment (22 and 26 in FIG. 4) was shown to contain a long, opentranslation reading frame when it was sequenced and the sequence datawas analyzed using the University of Wisconsin sequence analysisprogrammes.

Results of analysis of the nucleotide sequence of part of the region ofthe glucoamylase gene between two Eco RI sites within the Hind III-BglII fragment are shown in FIG. 6. This region contains the glucoamylasepromoter and the signal peptide sequence.

Within this fragment i.e. at nucleotides 97-102 is a "TATA box", namelythe familiar sequence ⁵ ' TATAAA³ ' which provides a site required bymany eukaryotic promoter regions for accurate initiation oftranscription (probably an RNA polymerase II binding site). Accordingly,the presence of at least a portion of the promoter region is confirmed.Further, it is predictable from analogy with other known promoterregions that all the functional essentials are likely to be containedwithin a sequence of about 1,000 bases in length and most likely withinthe first 200 -- bases upstream of the start codon for the coding regioni.e. nucleotides 206-208 or "ATG", the codon for methionine. Thus, thepromoter and transcript leader terminate at nucleotide 205. The identityof the beginning of the promoter region is less crucial although thepromoter region must contain the RNA polymerase II binding site and allother features required for its function. Thus, whereas the Eco RI-EcoRI sequence is believed to represent the entire promoter region of theglucoamylase gene, the fragment used in plasmid pGL2 contains thisfragment in the much larger Hind III-BamH I/Bgl II segment to ensurethat the entire promoter region is properly included in the resultantplasmid.

On the basis that the amino acid sequence of mature glucoamylase isknown (see Svensson et al, "Characterization of two forms ofglucoamylase from Aspergillus niger", Carlsberg Res. Commun, 47, 55-69(1982)), a nucleotide sequence of the signal peptide can be determinedaccurately. The signal peptide coding region of genes encoding secretedproteins is known to initiate with the methionine residue encoded by theATG codon. Determination of a sufficient initial portion of thenucleotide sequence beyond i.e. 3' of the ATG codon provides informationfrom which the amino acid sequence of that portion may be determined. Bycomparison of this amino acid sequence with the published amino acidsequence, the signal peptide can be identified as that portion of theglucoamylase gene which has no counterpart in the published sequencewith which it was compared. The glucoamylase signal peptide codingregion defined herein was previously confirmed using this method.

By the above methods, the Hind IlI-Bam HI/Bgl II fragment resulting fromSau 3A partial digestion and incorporated into pUC12 was confirmed tocontain the following features of the glucoamylase gene: an initial,perhaps non-relevant section, the promoter region, the signal peptidecoding region and the remaining portion of the coding region. Thisfragment, inserted into the pUC12 plasmid by scission with Hind III andBarn HI/Bgl II and ligation appears schematically in FIG. 4 as plasmidpGL1. This plasmid contains all of the features necessary forreplication and the like in order to remain selectable and replicable inE. Coli and selectable in A. niger.

B) Construction of Plasmid pGL2

Using pGL1 as a precursor, plasmid vector pGL2 can be formed as shown inFIG. 4. The restriction site BssH II which immediately follows the 3'end of the signal sequence 22, is utilized together with the unique SstI site following in order to insert the synthetic linker sequencesdefined in FIGS. 5A-5C herein. Thus, pGL1 is cleaved with both BssH IIand Sst I thereby removing the 5' portion of the structural glucoamylasegene 26 contained therein. Thereafter a selected one of the syntheticleader sequences A through C having been designed so as to be flanked byBssH II/Sst I compatible ends is inserted and ligated, therebygenerating plasmid pGL2. Depending on which of the three linkersequences is used i.e. A, B or C, the resultant plasmid will hereinafterbe identified as pGL2A, pGL2B or pGL2C, respectively.

The synthetic linker sequences identified herein are each equipped withunique Eco RV and Bgl II restriction sites into which a desired proteincoding region may be inserted. Once inserted, the resultant plasmid maybe used to transform a host e.g. A. niger, A. nidulans and the like. Thepresence of the promoter region and the signal peptide coding regionboth of which are recognized by the host, provide a means wherebyexpression of the protein coding region and secretion of the protein soexpressed is made possible.

EXAMPLE 4 Use of Plasmid pGL2

An example of the utility of the plasmid pGL2 is described below withreference to FIG. 7, which shows schematically the construction ofplasmid pGL2BIFN from pGL2B.

The plasmid pGL2B is prepared as described in general previously forpGL2 save that synthetic linker sequence "B" shown in FIG. 5B isinserted specifically. The reference numeral 24 has accordingly beenmodified in FIG. 7 to read "24B". In order to make available an openingin the vector pGL2B, the plasmid is cut with Eco RV at the site internalto linker 24B. The scission results in blunt ends which may be ligatedwith a fragment flanked by blunt ends using ligases known to be usefulfor this specific purpose.

In the embodiment depicted in FIG. 7, a fragment 28 containing thecoding region of human interferon 2 is inserted to Create pGL2BIFN.Specifically, a Dde I-Barn HI fragment 28 containing the coding regioncoding for human interfetch α2 was excised from plasmid pN5H8 (notshown) on the basis of the known sequence and restriction map of thisgene.

The plasmid pN5H8 combines known plasmid pAT153 with the interfetch geneat a Bam HI site. The interferon gene therein is described by Slocomb,et. al., "High level expression of an interferon α2 gene cloned in phageMl3mp7 and subsequent purification with a monoclonal antibody"Proceedings of the National Academy of Sciences, U.S.A., Vo. 79 pp5455-5459 (1982)

In order to anneal the sticky ends of the interferon fragment into thecut Eco RV site of pGL2B, the sticky Dde I and Barn HI ends are filledusing reverse transcriptase and ligated with an appropriate ligaseaccording to techniques standard in the art.

The advantage of selecting linker sequence B for insertion into pGL2 ismanifest from FIG. 8 which shows the reading frame of the interferon 2coding region and its relationship within the synthetic signal peptidesequence, in terms of nucleotide sequence and amino acid sequence, whereappropriate.

FIG. 8 shows the promoter region 5' of the signal sequence joined withthe glucoamylase signal peptide sequence beginning with the methioninecodon ATG and ending with the lysine codon AAG at 32. In fact, althoughthe signal peptide coding region extends one residue further i.e. to theCGC codon for arginine at 34, this latter residue is comprised by thesynthetic linker sequence engineered so as to compensate for the loss ofthe arginine residue during scission and ligation to insert the linkersequence. In this way, the genetic sequence of the signal remainsundisturbed.

In a similar manner, the linker sequence provides for insertion of theinterferon 2 coding region without altering the reading frame thereof.Cleavage of linker sequence B by Eco RV results in linker fragments B'and B" having blunt ends designated 36, 38, respectively. Excision ofthe interferon 2 coding region at Dde I site results, after filling inof the sticky ends created by the enzyme, in the desired nucleotidesequence without harming the sequence of that coding region. Ligation ofthe Eco RV-cleaved linker sequence with the interferon sequence filledat the Dde I site maintains the natural reading frame of the interferoncoding region as evidenced by the triplet codon state between the linkerportion B' and the interferon coding region. Had the linker A shown inFIG. 5A been chosen, which bears one less nucleotide than the linker B,the entire reading frame would have been shifted by one nucleotideresulting in a nonsense sequence. By selection of synthetic linker B,codons are made available between the signal peptide sequence and theinterferon coding region which do not alter the reading frame of thecoding region, when the blunt ended 1F2 fragment is oriented correctly.The correct orientation is selected by sequencing clones with insertsacross the ligation junction.

EXAMPLE 5 Expression and Secretion from A. nidulans Transformaned withpGL2BIFN

The plasmid pGL2BIFN was cotransformed i.e. with a plasmid containingArg B+ gene as described more fully in U.S. patent application Ser. No.678,578 filed Dec. 5, 1984 into an Arg B- strain of A. nidulans with aseparate plasmid containing an arg B selectable marker. Arg B+transformants were selected of which 18 of 20 contained 1-100 copies ofthe human interferon 2 coding region (as detected by Southern blotanalysis).

Several transformants were grown on starch medium to induce theglucoamylase promoter and the extracellular medium was assayed for human1F α2 using the CellTech 1F α2 assay kit.

All transformants exhibited some level of synthesis and secretion ofassayable protein. Two controls, the host strain (not transformed) andone arg B⁺ transformant with no detectable human 1F α2 DNA showed nodetectable synthesis of 1F α2 protein. In a separate experiment,transformation of A. niger, rather than A. nidulans, with pGL2BlFNusing, mutatis mutandis, the same procedure as described above,demonstrated the ability of A. niger to secrete 1F α2.

Thus, although the promoter and signal regions of pGL2BIFN are derivedfrom A. niger they are shown to be operative in both A. nidulans and A.niger.

In the present invention, use may be made of promoter regions other thanthe glucoamylase promoter region. Suitable for use are the promoterregions of the alcohol dehydrogenase I gene and the aldehydedehydrogenase gene, illustrated in FIG. 1.

EXAMPLE 6 Construction of Plasmid pALCAlS

For use with the present example, the alcA promoter was employed ascomprised within an 10.3 kb plasmid pDG6 deposited with ATCC within hostE. Coli JM83 under accession number 53169. A plasmid map of pDG6 isshown in FIG. 9, to which reference is now made, to illustrate use ofthe alcA promoter.

pDG6 comprises the promoter region 50 of the alcA gene as well as asmall 5' portion 52 of the alcA coding region 3' of the start codon,ligated to the endoglucanase structural gene 54. pDG6 further comprisesa multiple cloning site 56 downstream of the C. fimi endoglucanasecoding region 54.

To retrieve the alcA promoter region 50, pDG6 was cut with Pst I and XhoI removing the bulk of the endoglucanase coding region 54. In a secondstep, the linearized plasmid was resorted in one direction in acontrolled manner with exonuclease III (which will resect from XhoI butnot PstI-cut DNA ends) followed by tailoring with nuclease Sl. Theresection was timed so that the enzyme removed nucleotides to a position50 bases 5' of the alcA ATG codon, leaving the TATA box and messengerRNA start site intact.

Following resection, the vector 58 was religated (recircularized)creating vector 60 bearing Sal I-Xba I restriction sites immediatelydownstream of the promoter region 50. Cleavage of vector 60 with SalI/Xba I permits introduction of a signal peptide coding region at anappropriate location within the vector.

The particular signal peptide coding region employed in the presentexample was synthesized to reproduce a typical signal peptide codingregion identified according to standard procedures as described by G.Von Heijne in Eur. J. Biochem. 17-21, (1983). The synthetic signal wasengineered so as to provide a 5' flanking sequence complementary to aSal I cleavage site and a 3' flanking sequence enabling ligation withthe Xba I restriction sequence.

The sequence of the synthetic secretion signal 64 is reproduced below:##STR1##

The secretion signal per se begins with Met and ends with Ala (fourthoccurrence).

Once generated, the synthetic sequence acting as signal is cloned intothe Sal I-Xba I site of vector 62 resulting in plasmid pALCAlS whichcontains alcA promoter region 50, and synthetic peptide signal codingregion 64. That the signal peptide coding region is inserted upstream ofthe multiple cloning site 56 is significant in that the site 56 allowsfor cloning of a variety of protein coding segments within this plasmid.

Accordingly, pALCAlS constitutes a valuable embodiment of the presentinvention.

EXAMPLE 7 Construction of Plasmid pALCAlSIFN

As an example of the utility of pALCAlS, reference is made to FIG. 10showing creation of pALCAlSIFN 66. This plasmid 66 comprises thepromoter region 50 of the alcA gene and the synthetic signal peptidecoding region 64 both of which are derived from pALCAlS (FIG. 9). Inaddition, it contains the structural gene 28 coding for human interferonα2 derived from pGL2BIFN.

To obtain the protein encoding segment, pGL2BIFN is cleaved with Eco RIand partially cleaved with Bgl II (because of the presence of internalBgl II sites). Insertion of the protein coding region is accomplished bycleaving pALCAIS with Bam HI and Eco RI both of which are available inthe multiple cloning site 56 and ligating this cooing region therein,thereby creating pALCAlSIFN.

The nucleotide sequence of the resultant plasmid, from Hind III to EcoRI is shown in FIG. 11, indicating the sites of restriction endonucleasedigestion. It will be noted from sheet 3 of FIG. 11 that the 1Fα2 codingregion is in proper reading frame with the synthetic signal peptidecoding region.

EXAMPLE 8 Expression and Secretion from A. Nidulans Transformed withPlasmid pALCAlSIFN

The plasmid pALCAlSIFN prepared as described above was co-transformedwith A. nidulans to provide an arg B selectable marker, the arg B+transformants selected and checked for the presence of the humaninterferon α2 coding region, then grown on a threonine-containing mediumto induce the alcA promoter, all as described in example 3 above. Theextracellular medium was assayed for human IF-2 using Cell Tech IF 2assay kit. Eleven of twenty transformants showed secretion ofinterferon, induced in the presence of threonine, and repressed in thepresence of glucose.

EXAMPLE 9 pGL2CENDO

In accordance with the procedures described in the previous examples,there was constructed a vector plasmid designated pGL2CENDO, fromplasmid pGL2C, analagous to pGL2BIFN shown in FIG. 7, but containing theendoglucanase coding region in place of the interferon 2 coding region,and using the synthetic linker sequence "C" (FIG. 5C) in place of linkersequence "B". A Bam HI fragment containing the C. fimi endogluconasecoding region was inserted into the Bgl II site. A. nidulanstransformants were prepared with this vector plasmid, and showed starchregulated secretion of cellulase assayed as described in Example 1. Themap of vector plasmid pGL2CENDO is shown in FIG. 12 of the accompanyingdrawings, in which 70 denotes the endoglucanase coding region (anendoglucanase from Cellulomonas fimi, described in connection with FIG.2 and Example 1), 72 denotes the signal peptide coding region of theglucoamylase gene and 74 denotes the promoter region of the glucoamylasegene. The nucleotide sequence is shown in FIG. 13 and exemplifies thatuse of linker sequence C (FIG. 5C) retains the reading frame of thesignal peptide coding region 72 and the endoglucanase coding region 70.

EXAMPLE 10 Construction of Plasmid pALCAlSENDO

In accordance with the procedures described in the previous examples,there was constructed a vector plasmid designated pALCAlSENDO bycombining Eco RI-- linearized plasmid pALCAlS as described in example 5(FIG. 9) with an Eco RI fragment derived from plasmid pDG5B (see FIG. 2)(pDG5 with the orientation of the Hind III fragment reversed in pUC12)and containing the endoglucanase coding region. The map of pALCAlSENDOis shown in FIG. 14 and the nucleotide sequence of its pertinent regionis shown in FIG. 15. In these figures, the promoter region derived fromalc A is designated by numeral 50, the synthetic signal peptide codingregion is designated 64 and the endoglucanase coding region isdesignated by reference numeral 30.

EXAMPLE 11 Expression and Secretion from A. nidulans Transformed withpALCAlSENDO and pGL2CENDO

A. nidulans was co-transformed with an arg B+ selectable marker and theplasmid pALCAlSENDO or pGL2CENDO prepared as described above. Of theco-transformants several showed varying levels of secretion of cellulase(i.e. endoglucanase) as assayed on carboxymethylcellulose plates and themonoclonal antibody test systems as described in example 1. Both plasmidtransormants showed secretion which was controlled by the linkedpromoter. Plasmid pGL2CENDO was induced by starch and pALCAlSENDO wasinduced with threonine.

EXAMPLE 12 Expression and Secretion From A. niger Transformed withpGL2CENDO

A. niger was cotransformed with an arg B+ selectable marker and theplasmid pGL2CENDO. Several of the transformants showed varying levels ofsecretion of endoglucanase as assayed as described in example 1. Thissecretion was induced by the presence of starch in the medium.

Thus, the present invention provides a means for introducing astructural gene into a host which, when transformed, will secrete thedesired protein. Particularly useful plasmids for this purpose arepALCA1S and pGL2 (A, B or C).

Useful transformation vectors derived from these plasmids includepALCAlSIFN, pGL2BIFN, pALCAlSENDO and pGL2CENDO. Cultures of each ofthese plasmids are currently maintained in a permanently viable state atthe laboratories of Allelix Inc., 6850 Goreway Drive, Mississauga,Ontario, Canada. The plasmids will be maintained in this conditionthroughout the pendency of this patent application and, during thattime, will be made available to the Commissioner of Patents and TradeMarks at his request. Prior to issue of a patent on this application,applicant presently intends to deposit these plasmids with the ATCCdepository recognized under the Budapest Treaty. The accession numbersof the respective deposits will thereafter be inserted into the tableappearing below:

    ______________________________________                                        Plasmid   Host       Accession #                                                                              Deposit Date                                  ______________________________________                                        pDG6      E. Coli JM83                                                                             53169      June 7, 1985                                  pGL2A     "          53365      Dec. 16, 1985                                 pGL2B     "          53366      "                                             pGL2C     "          53367      "                                             pALCA1S   "          53368      "                                             pALCA1SENDO                                                                             "          53370      "                                             pALCA1SIFN                                                                              "          53369      "                                             pGL2B1FN  "          53371      "                                             pGL2CENDO "          98027      August 7, 1995                                pALCA1AHY "          53380      December 20, 1985                             ______________________________________                                    

What is claimed is:
 1. A DNA construct for use in transforming anAspergillus host to obtain expression therein of a polypeptide, said DNAconstruct comprising inducible promoter DNA for promoting transcriptionin Aspergillus and operably linked to DNA coding for said polypeptide toenable expression thereof in said Aspergillus host, said DNA coding forsaid polypeptide being foreign to said promoter DNA.
 2. The DNAconstruct according to claim 1, wherein said inducible promoter DNA isglucose-repressible.
 3. The DNA construct according to claim 1, whereinsaid inducible promoter DNA is starch inducible.
 4. The DNA constructaccording to claim 1, wherein said inducible promoter DNA is ethanolinducible.
 5. The DNA construct according to claim 1, wherein inductionof said inducible promoter DNA is mediated by the alcR gene.
 6. The DNAconstruct according to claim 1, wherein said inducible promoter DNA isthe glucoamylase promoter of Aspergillus niger.
 7. The DNA constructaccording to claim 1, wherein the coding DNA is foreign to saidAspergillus host.
 8. The DNA construct according to claim 1, and furthercomprising a DNA fragment encoding a promoter-inducer substance.
 9. TheDNA construct according to claim 1, wherein said Aspergillus host isAspergillus niger.
 10. The DNA construct according to claim 1, whereinsaid Aspergillus host is Aspergillus nidulans.
 11. A DNA constructaccording to claim 1, and further comprising a signal peptide codingregion operably linked to said DNA coding for said polypeptide forsection of said polypeptide.
 12. The DNA construct according to claim 8,wherein said DNA fragment encodes alcR.
 13. An Aspergillus strain whichupon culturing expresses a polypeptide as a result of having beentransformed by a DNA construct in which DNA coding for the polypeptideis linked operably to inducible promoter DNA for promoting transcriptionin Aspergillus, said DNA coding for said polypeptide being foreign tosaid promoter DNA.
 14. The Aspergillus strain according to claim 13,wherein said inducible promoter DNA is glucose-repressible.
 15. TheAspergillus strain according to claim 13, wherein said induciblepromoter DNA is starch inducible.
 16. The Aspergillus strain accordingto claim 13, wherein said inducible promoter DNA is ethanol inducible.17. The Aspergillus strain according to claim 13, containing alterationswithin regulatory genes which influence the expression of said DNAconstruct.
 18. The Aspergillus strain according to claim 13, whereininduction of said inducible promoter DNA is mediated by the alcR gene.19. The Aspergillus strain according to claim 13, wherein said induciblepromoter DNA comprises the alcA promoter native to Aspergillus nidulansoperably linked to the C. funi endogluconase coding region.
 20. TheAspergillus strain according to claim 13, wherein said strain is atransformed Aspergillus species selected from the group consisting ofAspergillus nidulans and Aspergillus niger.
 21. The Aspergillus strainaccording to claim 13, wherein the coding DNA is foreign to saidAspergillus strain.
 22. The Aspergillus strain according to claim 13,wherein said inducible promoter DNA comprises the alcA promoter nativeto Aspergillus nidulans.
 23. An aspergillus strain according to claim13, wherein said DNA construct comprises a signal peptide coding regionoperably linked to said DNA coding for said polypeptide for secretion ofsaid polypeptide.
 24. The Aspergillus strain according to claim 18,wherein multiple copies of said alcR gene are present in said host toincrease expression of said inducible promoter DNA.
 25. The Aspergillusstrain according to claim 22, which is an Aspergillus nigertransformant.
 26. A method for producing a polypeptide, said methodcomprising the step of culturing an Aspergillus strain transformed by aDNA construct in which DNA coding for a polypeptide is linked operablyto inducible promoter DNA for promoting transcription in Aspergillus,said DNA coding for said polypeptide being foreign to said promoter DNA.27. The method according to claim 26, wherein said inducible promoterDNA is glucose-repressible.
 28. The method according to claim 26,wherein said inducible promoter DNA is starch inducible.
 29. The methodaccording to claim 26, wherein said inducible promoter DNA is ethanolinducible.
 30. The method according to claim 26, wherein induction ofsaid inducible promoter DNA is mediated by the alcR gene.
 31. The methodaccording to claim 26, wherein said inducible promoter DNA comprises thealcA promoter native to Aspergillus nidulans operably linked to the C.funi endogluconase coding region.
 32. The method according to claim 26,wherein the strain is transformed Aspergillus species selected from thegroup consisting of Aspergillus nidulans and Aspergillus niger.
 33. Themethod according to claim 26, wherein the coding DNA is foreign to saidAspergillus strain.
 34. A method according to claim 26, wherein said DNAconstruct comprises a signal peptide coding region operably linked tosaid DNA coding for said polypeptide for secretion of said polypeptide.35. The method according to claim 30, wherein multiple copies of saidalcR gene are present in said host to increase expression of alcA andaldA genes.